The long-term goal of this research project is to understand the mechanisms underlying Ca2+-mediated signaling and the molecular basis for diseases associated with alterations in Ca2+ homeostasis. Extracellular Ca2+ ([Ca2+]o) has been proposed to function as a first messenger to trigger diverse cellular processes. Ca2+-sensing receptors (CaRs) represent a class of receptors that respond to changes in [Ca2+]o and activate multiple signaling pathways. By serving as the body's "thermostats" for [Ca2+]o, CaRs play a central role in the regulation of [Ca2+]o homeostasis and represent important therapeutic targets. A major barrier to advancing our understanding of the role of Ca2+ in regulating CaRs is the lack of adequate information about the location of their Ca2+-binding sites and the structural information of this class of membrane proteins. Obtaining site-specific Ca2+-binding affinities of naturally-occurring proteins is hampered by the complexities encountered in cooperative, multi-site systems. The delineation of the Ca2+-binding sites in the CaR and related proteins is further hindered by limitations of crystallization conditions, rapid off-rates owing to low Ca2+-binding affinities and the existence of multiple conformations that are in equilibrium with one another.The immediate goals of this proposal are to 1) probe Ca2+-binding sites in the Ca2+-sensing receptors and 2) verify our prediction of specific Ca2+-binding sites by correlating the site-specific and domain-specific Ca2+-binding information with the biological activity of the w.t. receptor in mammalian cells as well as receptors with mutations in these Ca2+-binding sites. Results from our proposed work will have a major impact on the understanding of the mechanisms underlying the biological activities carried out by Ca2+-modulated receptors. These proposed investigations will provide novel methods for identifying Ca2+- binding sites in the CaR and related proteins, thus overcoming the major obstacles encountered in visualizing Ca2+-binding sites with weak binding affinities. Success in identifying Ca2+-binding sites and clarifying how Ca2+ regulates the CaR will not only promote an understanding of how Ca2+ functions as an extracellular messenger, but will also provide insights into the molecular basis of the clinical disorders associated with this receptor. Our success in designing and engineering metal-binding sites into arbitrary proteins could also lead to new ways of developing valuable reagents for diagnostic tests and chemotherapy.